Ultra high throughput DNA sequencing system based on two-dimensional monolith mul

描述（由申请人提供）：本项目的主要目标是开发、实施和测试一种高效的DNA测序方法和仪器，该方法和仪器能够自动重测序和重新测序哺乳动物大小的基因组，具有小鼠草稿质量[Nature 420: 520,2002]，成本降低100倍。该系统将基于高度平行的CE分离和检测由凝胶基质固定的单模板分子（polonies）集落在二维单块多毛细管阵列（2D-MMCA）中产生的荧光标记的双脱氧核苷酸终止DNA延伸产物。成本的降低将来自于使用纳升体积反应和使用2D-MMCAs，与常用的高通量DNA机器相比，2D-MMCAs将CE分离和检测的吞吐量提高了至少两个数量级。我们将执行以下工作：开发技术平台，构建试验性55x55车道自动DNA测序仪，测序速度为1000 bp/秒，Q20读取长度为450bp，成本为0.007美元/kbp；对所提出的系统进行进一步开发，并引入技术和仪器变化，实现从头测序：优化polonie培养技术，获得3-5kb DNA片段的高效扩增和循环测序；建立一个试验性100 × 300车道DNA测序仪，能够以7500 bp/秒的速度自动生成原始测序数据，800 bp Q20读取长度，成本为0.002美元/kbp。演示该系统在10倍覆盖范围内对人类基因组进行从头测序的潜力，成本为10万至20万美元。

DESCRIPTION (provided by applicant): The main goal of the present project is a development, implementation and testing of a highly efficient method and an instrument for DNA sequencing capable of both automated re-sequencing and de-novo sequencing of mammalian size genomes with the mouse draft quality {Nature 420:520, 2002} at 100-fold reduced cost. The proposed system will be based on highly parallel CE separation and detection of fluorescently labeled dideoxynucleotide-terminated DNA extension product generated by gel matrix-immobilized colonies of single template molecules (polonies) in two dimensional monolith multi-capillary arrays (2D-MMCA). The cost reduction will come from both using nanoliter volume reactions and employing 2D-MMCAs which increase the throughput of the CE separation and detection by at least two orders of magnitude compared to commonly used high-throughput DNA machines. We will perform the following: Development of the technology platform, building of a pilot 55x55 lane automated DNA sequencer capable of sequencing 1,000 bp/second with 450bp Q20 read length at cost of $0.007/kbp, and demonstration of the production scale re-sequencing and automated generation of a genome sequence of the quality of the mouse draft genome at less than $100,000 per mammalian size genome Further development of the proposed system and introducing the technology and the instrumentation changes enabling de-novo sequencing: Optimization of polonie's cultivation technology and obtaining an efficient amplification and cycle sequencing of 3-5kb DNA fragments; Building a pilot 100x300 lane DNA sequencer capable of automated generation of raw sequencing data at 7500 bp/second with 800 bp Q20 read length at cost of $0.002/kbp. Demonstration of the system's potential for de-novo sequencing of human size genome at 10 fold coverage for $100,000 - $200,000.